First detection of kdr L1014F allele in Anopheles ziemanni and Anopheles pharoensis in Cameroon and distribution of the allele in members of the Anopheles gambiae complex

Background Knockdown resistance (kdr) is one of the primary resistance mechanisms present in anopheline species. Although this mutation is largely spread across the Anopheles gambiae s.l. members, its prevalence in other species is still not well documented. Methods The present study investigated the distribution and allelic frequencies of kdr in An. gambiae s.l., An. pharoensis, and An. ziemanni samples collected in 2022 and 2023 in nine sites spread across five ecogeographical settings in Cameroon. Members of the An. gambiae complex were identified molecularly by polymerase chain reaction (PCR). kdr L1014F and L1014S alleles were screened by PCR and confirmed by sequencing. Results An. gambiae (49.9%), An. coluzzii (36.5%), and An. arabiensis (13%) were identified, and the frequency of the kdr L1014F was high in both An. gambiae and An. coluzzii in all sites. The kdr L1014F allele was detected for the first time in 8 out of 14 An. ziemanni samples examined and in 5 out of 22 An. pharoensis samples examined. The kdr L1014S allele was scarce and found only in the heterozygote “RS” state in An. arabiensis and An. gambiae in Yangah and Santchou. Conclusions The present study sheds light on the rapid expansion of the kdr L1014F allele in malaria vectors in Cameroon and stresses the need for surveillance activities also targeting secondary malaria vectors to improve the control of malaria transmission. Graphical Abstract


Background
Malaria remains a major public health problem in Cameroon [1].In 2022, there were over six million malaria cases reported in health care centers across the country.It is estimated that 24% of the 25 million Cameroonians have at least one malaria attack each year [2].Disease incidence is estimated to vary between 100 and 196 per 1000 according to epidemiological records [1].Despite the frequent distribution of bed nets across the country, there has not been a significant decline of malaria [1].Among factors affecting vector control measure performance are the rapid expansion of insecticide resistance and the high diversity of vector populations, which display different feeding, resting, and biting behaviors [3].Studies characterizing resistance mechanisms in vector populations indicated a rapid increase of insecticide resistance in Anopheles gambiae s.l. and An.funestus with multiple resistance profiles [4][5][6][7].Recent studies also indicated a reduced level of insecticide susceptibility of several other anopheline species, including An. moucheti, An. coluzzii, An. nili, and An.rufipes to dichlorodiphenyl-trichloroethane (DDT) and pyrethroids [8][9][10].Apart from An. gambiae s.l. and An.funestus for which resistance mechanisms have been extensively explored, few studies characterizing resistance mechanisms in other anopheline species have been undertaken [9].
Different mechanisms including metabolic, cuticular, and target site mutations [e.g., knockdown resistance (kdr)] drive resistance to insecticides in mosquitoes [11].kdr mutations, among the most widely spread resistance mechanisms, consist in aminoacidic substitutions in the voltage-gated sodium channel (Vgsc) that reduce the binding and/or action of pyrethroids and DDT and, thus, result in a reduced susceptibility to these insecticides [12,13].This resistance mechanism is highly frequent in An. gambiae with two widespread resistance alleles: the L1014F allele widely distributed in West and Central Africa and the L1014S allele more frequent in Eastern Africa [14][15][16].However, there are still not enough data on the distribution of these alleles in other Anopheles species.The present study investigated the distribution of these alleles in An. gambiae, An. coluzzii, An. arabiensis, An. pharoensis, and An.ziemanni mosquitoes collected across Cameroon.
Mosquitoes were collected from nine locations belonging to five different ecogeographical areas in Cameroon (dry savanna, humid savanna, highlands, coastal, and forest) (Fig. 1 and Table 1) during the periods of September to November 2022 and June to August 2023 in the raining season, using different sampling methods, including Centers for Disease Control light traps, human landing catches, and Prokopack aspirators.Adult mosquitoes were identified morphologically using the identification keys of Gillies and Coetzee (1987) [17] and Gillies and De Meillon (1968) [18] and preserved in silica gel for molecular analyses.
DNA extraction was done with the JETFLEX Genomic DNA Purification Kit (Invitrogen by Thermo Fisher Scientific) following the manufacturer's guidelines.Members of the An.gambiae complex were identified using the rapid high-throughput SYBR green assay described by Chabi et al. (2019) [19] and/or using the protocol of Favia et al. (2001) [20].Some An. phaorensis and An.ziemanni samples were sequenced at cytochrome c oxidase subunit 1 (COI) loci for species confirmation [21].
A subset of mosquito species collected from each site were used for the screening of kdr alleles 1014L/S.Allelespecific polymerase chain reaction (AS-PCR) was used to detect L1014F (AS-PCR Agd3) and L1014S (AS-PCR Agd5) alleles as described by Verhaeghen et al. ( 2006) [22].Some samples of An. gambiae s.l., An. ziemanni, and An.pharoensis were later Sanger sequenced for the confirmation of the presence/absence of the mutation at the Microsynth Company (Germany).
After checking the quality of the chromatograms, we blasted the sequences (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi? PROGR AM= blast n& PAGE_ TYPE= Blast Searc h& LINK_ LOC= blast home) and aligned them in reverse and forward direction using ClustalW (https:// www.genome.jp/ tools-bin/ clust alw).The kdr L1014F and L1014S mutations were detected studying the picks of the chromatograms corresponding to the mutation sites.
Anopheles gambiae was recorded in almost all sites, while An.arabiensis was only found in Yangah together with An. pharoensis and An.ziemanni.In Mbanjock, Djohong and Dschang, only An. gambiae was found, whereas in Kribi, only An. coluzzi was registered.Both An. gambiae and An.coluzzii were found in sympatry in Mbalmayo, Yaoundé, and Douala.
The kdr allele L1014F was found at very high frequency in both An.gambiae (PQ000897) and An.coluzzii (PQ000899) in all sites, while only 2 An.arabiensis out of 59 were found with the allele (PQ000905) (Table 2).The kdr allele L1014S was scarce and detected only at the heterozygote "RS" state in An. arabiensis (PQ000906) and An.gambiae in Yangah and Santchou.One An. gambiae sample (PQ000898) was found with the double mutation L1014F/S (Table 2).It is noteworthy that the kdr allele L1014F was detected for the first time in An. ziemanni and An.pharoensis: out of the 14 An.ziemanni examined, 7 were found to be homozygotes "RR" (PQ000903) and 1 was heterozygote "RS" (PQ000901).Out of the 22 An.pharoensis examined, three were found to be homozygotes "RR" (PQ000907) and 2 were heterozygote "RS" (PQ000909) (Table 2).No kdr 1014S was detected in An. ziemanni and An.pharoensis.
The present study objective was to investigate the presence of kdr alleles 1014F/S and their frequencies in mosquito samples collected from different parts of Cameroon.The study indicated a high prevalence of the kdr allele L1014F in both An.gambiae and An.coluzzii in all study sites.This result was similar to studies conducted so far in Cameroon reporting a high frequency of the kdr resistance allele in members of the An.gambiae complex [4,[23][24][25].
Interestingly, the allele L1014F was also detected for the first time in both An.ziemanni and An.pharoensis.These species are considered as secondary malaria vectors in Cameroon owing to their low implication in  malaria transmission and their highly zoophilic and exophilic behavior [3,26].The presence of this mutation in these species could result from the high selective pressure induced using pesticides in agriculture.Indeed, the site of Yangah where An. ziemanni and An.pharoensis were sampled is a locality where rice, millet, and cotton are cultivated in large surfaces.The production of these crops requires the use of large quantities of pesticides [27,28].Although no bioassays were performed in the present study to evaluate the susceptibility of An. ziemanni and An.pharoensis to DDT and pyrethroids, previous studies conducted in the area and surrounding localities indicated a low susceptibility of local anopheline species to these insecticides [8,10,24].It should be important for future studies to explore the presence of other resistance mechanisms also in secondary vector species as a recent study indicated the implication of cuticular resistance in An. pharoensis samples resistant to DDT [10].

Conclusions
The spread of kdr alleles in other anopheline species is problematic for the use of pyrethroids in public health.Even though, during the last distribution campaign new-generation bed nets, pyrethroid-piperonyl butoxide (PBO) (Olyset Plus ® ), and interceptor ® G2 (IG2), combining pyrethroids with other active ingredients were distributed to combat resistant vector populations [1], the impact of this new control strategy is still awaited.The rapid expansion of resistance in vector populations must therefore continue to be the subject of particular attention, as it could compromise the control efforts implemented in the field.

Fig. 1 A
Fig. 1 A map of Cameroon showing the collection sites.The nine collection sites (black dots) are distributed in five of the six ecogeographical areas

Table 1
Descriptions of the study sites

Table 2
Distribution of the kdr alleles L1014F/S in anopheline species collected in different sites across Cameroon